Melanin-free Ink Research Articles

Caviar-like hydrogel beads containing different concentrations of NaCl and melanin-free squid ink

Different concentrations (0, 0.25, 0.5, 1 and 2%) of melanin-free ink (MFI) obtained from squid (Loligo vulgaris) discards was incorporated into alginate hydrogels containing different concentrations (3 and 5%) of NaCl to produce a blackish colored caviar-like product. Addition of MFI up to 1% into the caviar-like beads (CLB) solution increased physicochemical and sensorial properties but further (2%) addition significantly decreased sensorial properties such as odor, taste and liking scores. An increase in NaCl concentration in the bead formulation from 3 to 5% significantly increased the bulk density, MFI releasing rate, turbidity and hardness values of CLB, whereas L* color values decreased. CLB containing 3% NaCl were almost spherical shape compared to beads containing 5% NaCl which were tear shape. Addition of MFI into the CLB formulation did not affect the shape of beads. At 3% NaCl, the MFI and alginate were strongly attractive and so the MFI was better retained within the beads during storage. The CLB containing 3% NaCl and 1% MFI was the most preferred product for the hedonic sensory test and it could be an alternative to sturgeon caviar. Encapsulation of MFI in alginate beads as a colorant and bioactive compound could be a promising technique for caviar-like hydrogel bead production.

Potential of melanin free ink as antioxidant and its application for preserving and predicting shelf life of salted-boiled milkfish

Antioxidant could be extracted and isolated from squid inks. Squid ink in the form of melanin free ink (MFI) could be act as an electron donor which can stabilise free radicals in lipid oxidation. Studies were carried out to assess the antioxidant activity of squid inks converted into MFI in different dilution and to optimise the extraction conditions for the application of MFI as an anti-oxidative agent on fish product. Three different concentrations of MFI extracts i.e., 0%, 0.75% and 1.5% were applied on soft-boned milkfish at 10°C, 20°C and 30°C temperature storages for nine days, with observation every three days. The results showed the animism of the ink through chemical parameters, antibacterial effect, and sensory analysis. MFI at a concentration of 0.75% was revealed to be effective in prolonging cold storage shelf life of soft boned milkfish, which was confirmed through comprehensive sensory analysis evaluation and measurement of chemical changes including water, protein, lipid and ash content. It was also discovered that 0.75% MFI significantly prolonged the cold storage period of soft boned milkfish until six days, indicating that it may be developed to be a natural preservative for marine products.

Potential of melanin free ink as antioxidant and its application for preserving and predicting shelf life of salted-boiled milkfish

Antioxidant could be extracted and isolated from squid inks. Squid ink in the form of melanin free ink (MFI) could be act as an electron donor which can stabilise free radicals in lipid oxidation. Studies were carried out to assess the antioxidant activity of squid inks converted into MFI in different dilution and to optimise the extraction conditions for the application of MFI as an anti-oxidative agent on fish product. Three different concentrations of MFI extracts i.e., 0%, 0.75% and 1.5% were applied on soft-boned milkfish at 10°C, 20°C and 30°C temperature storages for nine days, with observation every three days. The results showed the animism of the ink through chemical parameters, antibacterial effect, and sensory analysis. MFI at a concentration of 0.75% was revealed to be effective in prolonging cold storage shelf life of soft boned milkfish, which was confirmed through comprehensive sensory analysis evaluation and measurement of chemical changes including water, protein, lipid and ash content. It was also discovered that 0.75% MFI significantly prolonged the cold storage period of soft boned milkfish until six days, indicating that it may be developed to be a natural preservative for marine products.

Effect of Squid Melanin-Free Ink and Pre-Emulsification on Properties and Stability of Surimi Gel Fortified with Seabass Oil during Refrigerated Storage

ABSTRACTThe effects of melanin-free ink (MFI) and pre-emulsification on gel properties and stability of bigeye snapper surimi gel fortified with seabass oil during refrigerated storage of 10 days were studied. Lipid oxidation as determined by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) of surimi gel increased as the level of seabass oil increased (P < 0.05). When MFI was incorporated into surimi gel, lower PV was obtained throughout the storage (P < 0.05). Addition of seabass oil pre-emulsified with soy protein isolate (SPI) in the presence of MFI yielded surimi gel with the highest breaking force and could improve oxidative stability during refrigerated storage (P < 0.05). Slight decrease in whiteness was found in surimi gel added with MFI, while those added with MFI along with pre-emulsified seabass oil showed increased whiteness (P < 0.05). Addition of MFI did not affect total viable count and psychrophilic bacterial count in surimi gels. Thus, the incorporation of pre-emulsified seabass oil prepared using SPI in conjunction with MFI could improve quality and oxidative stability of gel from bigeye snapper surimi.

Aryl Butenes Active against K562 Cells and Lacking Tyrosinase Inhibitory Activity as New Leads in the Treatment of Leukemia.

The inhibitory effects of four series of aryl butene derivatives, active against breast cancer, on the monophenolase activity of tyrosinase, in melanin-free ink from Sepia officinalis, have been studied. Hydroxytamoxifen 1, ferrociphenol 17 and several aryl butene analogs have shown strong antiproliferative activity on hormone-dependent and hormone-independent breast cancer cells and were evaluated in leukemia K562 cell proliferation. Their potential to induce skin depigmentation by evaluating their anti-tyrosinase activity was also estimated. In order to better rationalize the tyrosinase inhibitory action and the binding mode of the compounds, docking studies were carried out. Hydroxytamoxifen and some aryl butenes showed strong antiproliferative effects against K562 cells at 1 µM without showing tyrosinase inhibition. If phenolic compounds 16 and 17 showed the best antiproliferative activity on K562, Hydroxytamoxifen and compounds 5, 10, 20 and 21 have been identified as candidates for further development against chronic myeloid leukemia (CML), and are predicted to not induce depigmentation of the skin, a side effect encountered with imatinib, conventionally used for the treatment of CML.

Effect of Melanin Free Ink Extracted From Squid (Loligo sp.) on Proximate and Sensory Characteristics of Soft-Bone Milkfish (Chanos chanos) During Storage

Antioxidant could be extracted and isolated from squid inks. Squid ink in the form of melanin free ink (MFI) could be act as an electron donor which can stabilize free radicals in lipid oxidation. This study was carried out to assess the antioxidant activity of squid inks converted into MFI in different dilution and to optimize the extraction conditions for the application of MFI as an antioxidative agent on fish product. Three different types of MFI extracts i.e : pure squid ink, squid ink with 5 times dilution and squid ink with 10 times dilutions by using cooled ionized water (4°C). The ink was then centrifuged at 18.000 x g for 30 minutes at cooled centrifuge (4°C) followed by DPPH analysis. The results showed that the IC50 of MFI extracts were 2.84 ppm; 1.11 ppm and 0.34 ppm, respectively (p < 0.05). The results indicated that squid ink with 10 times dilution in extraction of MFI had the highest value in free radical inhibitory. Although the IC50 of three different dilutions are equally low, and are considered as very strong antioxidative agent, however, it showed that the MFI extracted from squid ink had the ability to prevent free radical

Effect of Melanin-Free Ink on Mechanical Properties and Yellow Discolouration of Protein Film from Washed Sardine Mince

The effect of the melanin-free ink (MFI) at different concentrations (0, 0.1, 0.2, 0.3 and 0.4 gL−1 of film forming solution (FFS)) on the properties and yellow discolouration of films from washed sardine mince was studied. Tensile strength (TS) of the film increased with increasing MFI concentration (P < 0.05). Conversely water vapour permeability (WVP) of films decreased as the concentration of MFI increased (P < 0.05). Fourier transform infrared (FTIR) spectra of films showed a slight shift to lower wavenumber of amide-B band of film added with MFI, indicating protein cross-linking. The microstructure showed slightly rough surface of the films when amount of MFI increased. However, cracks in the films were much decreased when MFI at higher levels was incorporated. Films added with MFI had lower thiobarbituric acid reactive substances (TBARS) value, indicating the lowered lipid oxidation. The addition of MFI also decreased the yellow discolouration but increased the transparency of film in a dose-dependent manner.

Application of melanin-free ink as a new antioxidative gel enhancer in sardine surimi gel.

The squid ink that is discarded as waste during processing can be effectively utilised as a gel enhancer in surimi gels, especially those prepared from dark-fleshed fish which have poor gel properties. It also acts as an antioxidant, inhibiting lipid oxidation. This investigation aimed to study the effect of melanin-free ink (MFI) from splendid squid (Loligo formosana) on properties and oxidative stability of surimi gel from sardine (Sardinella albella). MFI (0-0.1 g kg(-1) surimi) increased the breaking force and deformation of sardine surimi gel in a dose-dependent manner (P < 0.05). The addition of MFI had no effect on whiteness of surimi gels (P > 0.05). The expressible moisture content of gels decreased as the levels of MFI increased (P < 0.05). Based on a microstructure study, gel added with MFI at a level of 0.08 g kg(-1) surimi was denser and finer than that of the control (without MFI). Surimi gels with MFI had lower peroxide values, thiobarbituric acid reactive substances, nonanal and 2-decenal. MFI could improve the properties of sardine surimi gel. Additionally, it was able to prevent lipid oxidation in surimi gels during refrigerated storage.

Antioxidative activity of melanin-free ink from splendid squid (Loligo formosana)

Splendid squid (Loligo formosana) is the species available in Thailand and used for local consumption and export. The squid ink is discarded as by-product which can be used as a source of active compounds. Therefore, the aim of this study was to investigate the antioxidant activity of squid ink after melanin removal. The antioxidative activities of melanin-free ink (MFI) from splendid squid were investigated using different in vitro assays. Antioxidative activity of MFI was also determined in different model systems. Thermal stability at 90°C was tested. MFI was subjected to ultrafiltration, and fractions with various molecular weight (MW) ranges were analyzed for antioxidative activities. MFI had DPPH, ABTS radical scavenging activities, and ferric reducing antioxidant power (FRAP) of 179.6 ± 2.1, 957.8 ± 89.3, and 171.2 ± 7.3 μmol TE/g protein, respectively. It also possessed metal chelating activity of 4.0 ± 1.2 μmol EE/g protein. MFI at 500 mg/L could prevent the oxidation of β-carotene-linoleic acid system, but its efficacy was lower than butylated hydroxyl anisole (BHA) at 200 mg/L (p 10 KDa), the fractions with MW less than 3 KDa showed the highest antioxidative activities (p < 0.05). MFI, especially its fraction with MW < 3 KDa, from splendid squid could therefore serve as natural antioxidant to retard lipid oxidation in food products.

Ink is a conspecific alarm cue in the Caribbean reef squid, Sepioteuthis sepioidea

Animals often respond to attacks from predators with behaviors directed at the predator that increase the probability of escape. Some animals also respond to attacks with behaviors that release signals or cues that are detected by conspecifics and enhance escape. Cephalopods, with their complex nervous systems, produce many behaviors, one of which is ejecting ink when attacked by predators. Ink is thought to enhance the ability of the inking cephalopod to escape from the predator, which is a reasonable idea but is lacking experimental support and description of mechanisms. Inking might also serve as an alarm cue, evoking alarm responses in conspecifics. The aim of our study was to determine if ink of the Caribbean reef squid, Sepioteuthis sepioidea, is a conspecific alarm cue. We studied freely behaving animals in the laboratory to determine if they produced alarm or escape behavior in response to ink. We observed that squid responded to conspecific ink introduced into their aquarium with deimatic behaviors containing chromatophore elements (‘eye spots’, ‘all brown’, and ‘flashing’), cryptic behaviors with chromatophore elements (‘clear’, ‘plaid’, ‘dark arms’), cryptic behaviors with postural responses (‘sargassum’, ‘bad hair’), and protean behavior (‘fin movement’, ‘inking’, ‘jetting’). Squid also responded to ink introduced into an adjacent aquarium within view, thus demonstrating that vision can contribute to the alarm response. Melanin-free ink did not evoke a significant alarm response. This is the first experimental demonstration that squid respond to ink of conspecifics as a visual alarm cue. Ink may also provide a chemical alarm cue for S. sepioidea, though further behavioral studies are needed to explore this.

Physicochemical characterization of Sepia officinalis ink and the effects of storage conditions on the coagulation process

Sepia officinalis produces a dark ink constituted of a suspension of melanin granules in a viscous colourless medium showing a large variability in composition. The examination of the spectra obtained by scanning electron microscopy of crude and the melanin-free ink showed slight variation in elemental composition related to the elimination of melanin substance after centrifugation. Ink elemental content varied also depending on the period of sampling. Temperature, light and oxygen can be considered as coagulation factors. Temperature around ambient temperature (e.g. 30°C) gave strong coagulation, while lower temperature (2–4°C), lack of oxygen and darkness greatly inhibited the ink coagulation process. Moreover, we showed that hydrogen peroxide activated the ink coagulation process and the coagulation rate depends on the amount of H2O2 added. Heat treatment (100°C for 5 minutes) of ink inhibited the coagulation. Interestingly, the addition of an adequate volume of fresh melanin-free ink to the heated sample activated significantly the coagulation process.

Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid Ommastrephes bartrami by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy

A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage information and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid-fucose (GlcA-Fuc) disaccharide repeat in the main chain and a N-acetylgalactosamine (GalNAc) branch at Fuc position 3: -[3GlcAbeta1-4(GalNAcalpha1-3)Fucalpha1](n)-. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment. In the negative-ion tandem mass spectrometric analysis, the unique (0,2)A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position-3 of the disaccharide repeat.

Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase

The melanin-free ink of the cephalopod Sepia officinalis is shown to contain a heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells. Gel filtration chromatography indicated that the toxic component was concentrated in those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the highest tyrosinase activity. SDS–PAGE analysis of the active fractions displayed a single major band migrating at an approximate molecular weight of 100 kDa, identical with that of the single tyrosinase band in the melanin-free ink. These data unambiguously demonstrated the identity of the toxic component with tyrosinase. Treatment of purified Sepia as well as of mushroom tyrosinase with an immobilized version of proteinase K resulted in a parallel loss of tyrosinase activity and cytotoxicity. Sepia apotyrosinase was ineffective in inducing cytotoxicity in PC12 cells. Purified Sepia tyrosinase was found to induce a significant increase in caspase 3 activity in PC12 cells, leading eventually to an irreversible apoptotic process. Overall, these results disclose a hitherto unrecognized property of tyrosinase that may lead to a reappraisal of its biological significance beyond that of a mere pigment producing enzyme.